Transfusion of red blood cells (RBCs) is a life saving maneuver in acute trauma, a life sustaining treatment for diseases of the bone marrow and hemoglobinopathies, and an important component of medical support for a variety of different pathologies. Generation of alloantibodies against donor RBCs can be a major impediment to transfusion therapy, especially in patients who require chronic transfusion. Although incompatible transfusion is strictly avoided, because of the risks of hemolysis, hemolysis is not the inevitable outcome of incompatible transfusion. On the contrary, many incompatible transfusion are given with no signs or symptoms of hemolysis. Likewise, up to 1/1000 healthy blood donors have anti-RBC autoantibodies. Thus, it appears that there are biological mechanisms in place by which host tissues can avoid destruction from their own antibodies or alloantibodies. It is only poorly understood why some transfusions do not result in hemolysis, despite antibody coating of donor RBCs. We have developed several murine models of incompatible RBC transfusion, using authentic human blood group antigens, in which incompatible transfusions do not hemolyze. In some cases, the offending antigen is lost form the RBC without damaging the RBC, which then circulates normally. This phenomenon (called antigen-loss) has been well described in humans for a number of blood group antigens, but only very little mechanistic understanding has been generated. To the best of our knowledge, we have described the only model of antigen loss from RBCs. We have likewise described a second model in which an incompatible transfusion leads to the clearance of most RBCs; however, the RBCs that survive appear to represent a distinct population that is resistant to normal hemolytic mechanisms. This same biology can be observed in humans who have persistent DAT positive donor RBCs in circulation after a hemolytic transfusion reaction. As above, to the best of our knowledge, we have described the only animal model of this process. In this grant, we propose a hypothesis driven elucidation of the mechanisms of both antigen-loss and hemolysis resistance. These findings have potential relevance not only to the biology of antibody binding RBCs, but more broadly to any process in which an antibody is bound directly to a tissue. RBCs give a unique advantage in that they neither synthesize new protein nor undergo division, providing a stable substrate upon which to analyze protein and cellular changes during the process of antibody binding and subsequent biologies (e.g. complement activation, Fc gamma receptor ligation, etc.) We propose specific efforts to maintain the tractable nature of murine systems while progressively humanizing the models, including human RBC antigens, human Fc gamma receptors, and humanized antibodies. We propose 3 specific aims. Specific Aim 1: Molecular and Cellular Mechanisms of Non-Hemolytic Antigen-Loss. Specific Aim 2: Molecular and Cellular Mechanisms of Hemolysis Resistance. Specific Aim 3: Effects of Murine and Human IgG Subtype on Antigen-Loss and Hemolysis Resistance.